This invention relates to a method for the derivitization of oligosaccharides to form synthetic N-linked glycoconjugates under conditions that maintain the .beta.-anomeric configuration.
In general, carbohydrates are attached to various conjugates (e.g., proteins and lipids) by either N(nitrogen)-glycosidic or O(oxygen)-glycosidic linkages. Most animal glycoproteins contain oligosaccharides that are linked to a polypeptide backbone by a N-glycosidic linkage between N-acetyl-glucosamine (GlcNAc) and asparagine (Asn). The nitrogen glycosidic linkage between the reducing terminal monosaccharide (pyranose form) and asparagine in glycoproteins is in the .beta.-anomeric configuration as shown by Formula I, below. ##STR1##
Oligosaccharides with a free reducing terminus can be isolated from a variety of plant and animal sources. In addition, oligosaccharides can be released from glycoproteins by chemical or enzymatic methods. These saccharides also have a reducing terminal monosaccharide residue, typically GlcNAc or GalNAc (N-acetyl-galactosamine).
Derivatives of these oligosaccharides are useful in basic research activities concerning the function of the carbohydrate moeities of naturally occurring glycoconjugates, in clinical research and diagnostic medicine and in clinical pharmacology and therapeutics. The following list illustrates these useful derivatives.
1) Biotin conjugates of oligosaccharides. PA0 2) Fluorescent conjugates of oligosaccharides. PA0 3) Lipid conjugates of oligosaccharides. PA0 4) Peptide conjugates of oligosaccharides. PA0 5) Amino-acid conjugates of oligosaccharides. PA0 6) Immobilized oligosaccharide to solid support (e.g. agarose gel columns, silicon chips, Petri dishes etc.). PA0 7) Drug conjugates of oligosaccharides. PA0 8) Chromophore conjugates of oligosaccharides. PA0 9) 1-N-Protected glycosylamine derivatives, e.g. with carbobenzoxy (CBZ) or 9-fluorenylmethoxycarbonyl (FMOC) protecting groups.
Glycosylamines also are valuable intermediates in the synthesis of N-nucleosides, glycosylthioureas and glycosylamino heterocycles of biological and pharmaceutical interest. See, e.g., Carbohydr. Res. 188, 35-44 (1989), and references cited therein.
It would be desirable to make these oligosaccharide derivatives such that the linkage between the asparagine and the reducing terminal GlcNAc (i.e. GlcNAc.fwdarw.Asn) which occurs in glycoproteins as in Formula I is preserved. That is, it would be desirable to maintain the .beta.-anomeric configuration, pyranose form and carbonyl and methylene components of the asparagine. Formula II illustrates the nature of the GlcNAc.fwdarw.Asn linkage in glycoproteins; Formula III shows the chemical form of illustrative derivatives which thus would preserve the characteristics of the GlcNAc.fwdarw.Asn linkage. ##STR2##
A number of published methods for derivatizing oligosaccharides are available but they do not preserve all the aforesaid desired characteristics of the GlcNAc.fwdarw.Asn linkage.
One prior method of oligosaccharide derivatization involves reductive amination as described, for example, by Stowell and Lee, Adv. Carbohydr. Chem. and Biochem. 37, 225-279 (1980), especially pg. 245. However, the described techniques do not preserve the pyranose form and the anomeric centre of the reducing terminal monosaccharide, nor the carbonyl and methylene groups of asparagine as can be seen from the following two illustrative reaction schemes: ##STR3##
Another prior method of oligosaccharide derivatization involves formation of glycoconjugates by direct derivatization of glycosylamines as illustrated in PCT Inter. Pat. Appln. WO 88/04323, published Jun. 16, 1988. Although these techniques preserve the pyranose form of the reducing GlcNAc, they do not necessarily preserve the .beta.-anomeric configuration (a mixture of products is obtained) nor the carbonyl and methylene group of the asparagine as seen from the following illustrative product formula IV. Moreover, the methodology is applicable only to N-linked oligosaccharides attached to glycoproteins and, thereby, is of limited use. ##STR4##